MYOD represses gene expression from non-Ebox motifs

We report on the identification of a previously unrecognized property of MYOD as repressor of gene expression, via E-box-independent chromatin binding, during the process of somatic cell trans-differentiation into skeletal muscle. When ectopically expressed in proliferating human fibroblasts or endogenously induced in activated muscle stem cells (MuSCs) MYOD was detected at accessible regulatory elements of expressed genes, invariably leading to reduced chromatin accessibility and gene repression. At variance with conventional E-box-driven increased chromatin accessibility and H3K27 acetylation at previously silent loci of MYOD-activated genes, MYOD-mediated chromatin compaction and repression of transcription was associated with high occurrence of non-E-box motifs, did not lead to reduced levels of H3K27ac and coincided with reduced levels of H4 acetyl-methyl lysine modification (Kacme). Using MYOD mutants we dissected the molecular mechanism of MYOD-mediated repression, whereby repression of mitogen- and growth factor-responsive genes occurred via promoter binding, which requires a conserved domain within the first helix; conversely, repression of cell-of-origin/alternative lineage genes occurred via binding and decommissioning of distal regulatory elements, such as super-enhancers (SE), it required either the N-terminal activation domain or the two chromatin-remodeling domains, and it coincided with reduced strength of CTCF-mediated chromatin interactions. These data extend MYOD biological properties beyond the current dogma that restricts MYOD function to a monotone transcriptional activator. They also reveal an unprecedented functional versatility arising from alternative chromatin recruitment through E-box or non-E-box sequences, whereby genetic determinants dictate differential usage of MYOD functional domains.